Since the discovery of the mouse mammary tumor virus (MVTV) over three decades ago, this virus has been the subject of intensive in vivo investigation because of its role in the neoplastic transformation of the mammary gland of the mouse. However, no adequate in vitro method for the production of this virus is available. Viral preparations are routinely obtained from tumor tissue or from the milk of infected mice; thus attempts at purification and biochemical analysis are hampered by the presence of tissue contaminants. Further, there is no rapid in vivo assay, and no in vitro assay of MTV infectivity has yet been developed.' As a result, comparatively little is known about the molecular biology or antigenicity of MTV. Recently, an immunologic assay for MTV has been developed in our laboratories.2 The usefulness of this technique, which detects MTV antigenicity, has already been demonstrated in our in vitro studies.3, 4 This has prompted us to develop another in vitro assay for MTV, which measures virus production. In the technique described herein, MTV is labeled with radioisotope in organ culture and purified from the cell-free culture supernatants by density gradient centrifugation after the precipitation of contaminants with rabbit antisera. To date, we have used the technique to detect the production of MTV in organ cultures of MTV-infected normal mammary gland and mammary tumor tissue. We are currently using the procedure only as a qualitative assay, but it is subject to quantitation. Materials and Methods.-Tissues: All organ-cultured tissues were primary explants of mammary tissue obtained from isologous BALB/c (MTV-free) or BALB/cfC3H (MTVinfected) female mice from the colony of the Cancer Research Genetics Laboratory. MTV-free and MTV-infected normal tissues were obtained from the mammary parenchyma of pregnant BALB/c or BALB/cfC3H females, respectively. MTV-infected mammary tumor tissue was obtained from spontaneous tumors which developed in multiparous BALB/cfC3H females. A transplanted BALB/c mammary tumor was used as the source of MTV-free tumor tissue for control experiments; this tumor had developed in hyperplastic mammary tissue during prolonged hormonal stimulation and was kindly supplied by Mr. Dan Medina. Culture methods: Tumors were removed from the hosts and minced into I-mm' cubes. These explants were transferred by pipette to hanging-drop dishes5 or, in some experiments, onto rafts of lens paper in plastic organ culture dishes (Falcon Plastics, Los Angeles).6 Culture medium was defined MB752/1 (Waymouth's,7 Grand Island Biological Co.), with added cortisol succinate, 20 y/ml (Solucortef,? The Upjohn Co.), penicillin, 64 ug/ml, 10% fetal calf serum, and 2-20 ,c/ml H3-uridine. The cultures were incubated 5 days in the presence of a humidified mixture of 95% 02 and 5% CO2 at 37?C. The medium was changed daily and saved for analysis of virus content. Each sample consisted of